Peptide and process for preparing same from frog skin

ABSTRACT

A novel octapeptide is obtained by extracting the skin of an African frog (Xenopus laevis pipidae) with a lower alcohol, removing the solvent from the extract and subjecting the residue, in an optional order, to strongly acidic ion exchange resin chromatography, strongly basic ion exchange resin chromatography, gel filtration and droplet counter current partition chromatography.

United States Patent [191 Tachibana et al.

[ PEPTIDE AND PROCESS FOR PREPARING SAME FROM FROG SKIN [75] Inventors: Shinro Tachibana, Narashino;

Kengo Araki, Niiza, both of Japan [73] Assignee: Eisai Co., Ltd., Tokyo, Japan [22] Filed: Aug. 8, 1973 [21] AppL No.: 386,538

[30] Foreign Application Priority Data Aug. 18, 1972 Japan 47-82103 [52] US. Cl 260/112.5; 424/177 [51] Int. Cl. C071: 103/52; C07g 7/00; A6lk 27/00 [58] Field of Search 260/1125; 424/177 [56] References Cited OTHER PUBLICATIONS Anastasi et al., Brit. J. Pharmacol., 38, 22l-228 (1970).

[4 1 May 13, 1975 Anastasi et al., Arch. Biochem. Biophys, 108, 341-348 (1964).

Primary E.raminerLewis Gotts Assistant ExaminerReginald J. Suyat Attorney, Agent, or FirmWoodhams, Blanchard & Flynn [57] ABSTRACT 1 Claim, No Drawings PEPTIDE AND PROCESS FOR PREPARING SAME FROM FROG SKIN BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a process for preparing from the skin of an African frog a novel octapeptide having a contracting activity on a strip of fundus from a rat stomach and guinea-pig ileum in vitro and a hypotensive activity on rats.

2. Description of the Prior Art Valuable Chinese medicines represented by a toad poison, toad-cake, have been prepared from frogs from ancient times. Steroid compounds, histamine compounds, adrenaline compounds and serotonin compounds have heretofore been isolated from frog skins.

It has recently been found that in addition to these compounds, peptides having various biological and pharmacological activities are present in frog skins. As such peptides, there are known not only a group of bradykinins and derivatives thereof, but also caerulein having a strong contracting activity on gall bladder (Arch. Biochem. Biophys, 125, 57 (1968)), phyllomedisin having a hypotensive activity (Experientia, 26, 282 1970)), ranatensin having both a contracting activity on smooth muscle and a hypertensive activity (Fedr. Proc., 29, 282 (1970)), and alytesin and bombesin having a contracting activity on smooth muscle and a hypertensive activity (Experientia, 27, l66 (1971 SUMMARY OF THE INVENTION We have discovered that a novel peptide substance can be obtained from the skin of African frog (Xenopus laevis pipidae). We have further discovered that this substance has a contracting activity on rat stomach strip and a hypotensive activity.

In practicing the process of this invention, the skin of African frog is finely cut and then is extracted with a lower alcohol (ROH in which R is alkyl of 1 to 4 carbon atoms). Preferred lower alcohols include methanol, ethanol, isopropanol and the like. It is preferred that a very small amount, in the range of 0.01 to 1 percent by weight, based on the weight of the lower alcohol, of an aqueous solution of trichloroacetic acid is added to such lower alcohol. Trichloroacetic acid inhibits activities of enzymes and prevents decomposition of the intended product peptide of this invention. The solvent is then removed from the extract, and the residue is subjected, in an optional order, to (1) strongly acidic ion exchange resin chromatography, (2) strongly basic ion exchange resin chromatography, (3) gel filtration and (4) droplet countercurrent partition chromatography. There is obtained a fraction having a contracting activity on rat stomach strip and a hypotensive activity on rats. One or more of the foregoing treatments 1 to (4) may be repeated several times according to need. The order or sequence of these four treatments is not critical. These treatments can be carried out in various orders or sequences with satisfactory results.

As the strongly acidic ion exchange resin, there can be employed, for example SE-Sephadex (trademark for the product of Pharmacia Co.), SP-Sephadex (trademark for the product of Pharmacia Co.), Dowex 50W (trademark for the product of Dow Chemical Co.), Amberlite CG-l (trademark for the product of Rohm & Haas Co.) and the like. As the strongly basic ion exchange resin, there can be employed, for example, QAE-Sephadex (trademark for the product of Pharmacia Co.), Dowex l or 2 (trademark for the product of Dow Chemical Co.), Amberlite CG 400 (trademark for the product of Rohm & Haas Co.) and the like. As the gel filter, there can be employed, for me ample, Sephadex G25 (trademark for the product of Pharmacia Co), Bio-Gel P4 (trademark for the product of Bio-Laboratories Co.) and the like.

An example of a suitable droplet counter-current partition chromatography is as follows. Employing a liquid composed of n-butanolzacetic acid:water (4:1:5 parts by volume), a stationary liquid phase is formed as the upper layer and the substance to be purified is charged in the dissolved state into the lower layer of the column. The resulting fraction having a contracting activity on rat stomach strip is dried according to need. Thus is obtained the peptide product of this invention.

As a result of amino-acid analysis of the resulting peptide product, it was confirmed that the peptide product contains 1 mole equivalent of each of proline, glycine, glutamic acid, lysine, arginine, isoleucine and leucine. Tryptophan was also determined by the optical density at 280 mu. Thus, the peptide product consists of 8 amino acids.

The structure of the peptide of this invention is analyzed as follows:

1. Chymotrypsin Cleavage:

When the dansylated (dimethylaminonaphthalenesulfonylated) peptide (D-P) is subjected to chymotrypsin degradation, a dansyl-decomposed peptide (Cl is formed. As a result of amino acid analysis, it is found that C-l has a composition of lysine (l), arginine l glutamic acid (1), proline (1), glycine l) and tryptophan (1), Accordingly, in view of the substrate specificity of chymotrypsin, it is seen that the C-terminal amino acid of C l is composed of tryptophan.

2. Trypsin Cleavage:

When D-P is subjected to an action of trypsin, a dansyl-decomposed peptide (T-l is formed. As a result of amino acid analysis, it is found that T-l has a composition of lysine (1), arginine (l), glycine (l), and glutamic acid (1). In view of the substrate specificity of trypsin, it is seen that the Cterminal amino acid of T-l is composed of arginine.

3. Papain Cleavage:

When D-P is subjected to an action of papain, a dansyl-decomposed peptide (P-l is formed. As a result of amino acid analysis, it is seen that P-l has a composition of lysine (l), glutamic acid (1) and glycine (1 4. Degradation of C1 by Carboxypeptidase:

Cl is decomposed by carboxypeptidase A, and the reaction liquid is directly analyzed by an amino acid analyzer. As a result, only the presence of tryptophan is detected. The remaining reaction liquor is subjected to an action ofcarboxypeptidase B and the reaction liquor is directly analyzed by an amino acid analyzer. As a result, it is found that any amino acid other than tryptophan is not released. When the above results are considered in the light of the amino acid compositions of T-1 and Pl, it is seen that the penultimate amino acid positioned before tryptophan is proline.

5. Birch reduction:

The peptide is subjected to Brich reduction and the resulting reaction liquor is subjected to amino acid analysis. As a result arginine is found to have been dras 3 4 tically reduced. In view of this fact it is seen that the threshold dose 2 10 ng/ml amino acid positioned before proline is arginine. dose-response: not observed (one relaxed and then When the results described in (l) to (5) above are contracted) considered collectively. it is recognized that the pep- 3. Hypertensive Activity on Rats tide has an amino acid arrangement of (lysineglycine- 5 threshold dose 1.2 ug/Kg glutamic acid).arginineproline.tryptophan.(isoleucine tachyphylaxis: observed leucine). As regards the parenthesized portions, the Accordingly, it is seen that the peptide of this invenamino acid arrangement cannot be determined from tion is a valuable substance which possesses hypotenthe foregoing results. sive and gastrointestinal movement-promoting proper- 6. Analysis of C-Termina] Fragment: l ties. D-P is digested with chymotrypsin and dansylated to Further. the peptide of this invention has the followseparate dansylated (isoleucine leucine) (C2). Then. ing physical properties: C2 is hydrolyzed. As a result. the presence of da Rf values of dansylated peptide for thin layer chrosylated isoleucine is detected. The peptide is further graphy SiliCZl g subjected to an action of carboxypeptidase A. and the l Rf I -3 I yst m methylacetatefisoreaction liquid is directly analyzed by an amino acid anpr p nolramm ni 91714) alyzer, Leucine is detected and only a very minute Rfl 0.27 (SOlVCflI system n-butanol:acetic acid1waamount of isoleucine is detected. From this result it is {er 41115) een that th sequence f (L2 i i l i l i This invention will now be further described by refer- (free f rm), ence to the following illustrative example, in which the 7. Analy i f NTe i 1 A i A id; term active fraction means a fraction having a con- D-P is hydrolyzed at 100C. for 16 hours with 6N hytrading i i y 011 a IOm Ch trip. drochloric acid, and the dansylated amino acid is deter- EXAMPLE l mined and it is found that the dansylated amino acid is edansylated-lysine. Accordingly. it is seen that the N- 2 1 0f methanol Containing 5 of a 6 q termi i b]()cked D P i h d l d i 1N Caustic ous solution of trichloroacetic acid was added to fresh oda d all ed in Stand tiI] at 27C f 75 hours skins of African frogs. and the skins were allowed to Th i i uli d i h acetic id dansylated d stand still in a refrigerator maintained at about -20C. hydrolyzed with hydrochloric acid. As a result of idenfor a week- The resulting extract was Separated from ifl i f h d l d amino id i i f d that 30 the solid residue and then the extract was concentrated in addition to e-dansylated-lysine. dansylated glutamic under reduced W and drifid 10 Obtain Solids acid is present. Accordingly. it is seen that the N- g)- terminal amino acid is composed of pyroglutamic acid. A Column hm'ing diameter of 3 Cm and a 8. Analysis of C-terminal Amino Acid of P-l: length of 60 Cm was Packed with SE-Sephadex (3'25 P-l has an amino acid arrangement of pyroglutamic Well-bufferized with M ammonium formate P acidv (lysinelglyciney The C t i amine acid f 7.5 The solids were dissolved in 20 ml of 0.001 N for- P-] is determined by the tritium labelling method (Biomic acid and the solution was Charged in the columnchemical and Biophysical Research Communication, The gradient elution was efffiflfid h Ihfi use f 2 l 43. I334 l97l As a result, it is confirmed that the of water and l of ammonium formate P C i amino id i Composed fl i Th 1 6.5) at an elutlon rate of 60 ml/hr and at a unit fraction is found to have an amino arrangement of pyro volume Of ACtiVfi fractions t0 fracglutamic acidglycinelysine. tions) were collected and lyophilized.

F [hfl foregoing results, i i f d h {hg A second column having an inner diameter of 4.5 cm tide of this invention has the following structure: and a length of 9 cm was packfid i Sephadex py oglutamic ackiglycine lysine arginine proline well-buffcrlzed Wlth N acetic acid. The yp h i l i l i product was dissolved in 5 ml of 0.) N acetic acid and The following figure shows the attack sites of each the Solution was Charged in the Column- The penzyme to the dansylated peptide on the above menmerit EIUtiOl'l '13s effected the use Qf ht: Same SOitioned procedure for tructural dctcrminati n V6! at an elutlon rate Of and at a unit fraction volume of 3 ml. Active fractions (45th to th fractions) were collected and lyophilized. This lyophilized Y Y Y 'B 'Q'P 'Pf Leu'OH product was dissolved in 1 ml of 0.00l N aqueous ammonia and the solution was charged in a third column i 55 having a diameter of 0.9 cm and a length of 50 cm, in t which was charged QAE-Sephadex (A25) bufi'erized sufficiently with 0.00] N aqueous ammonia. The develc- 1 as c 2 j opment elution was effected with the use of 0.001 N aqueous ammonia at an elution rate of 12 ml/hr and a r e tidtlse A unit fraction volume of 3 ml. Active fractions (8th to The peptide of this invention has the following pharl5th fractions) were collected and lyophilized: macological activities at animal tests: The lyophilized product was purified by droplet l. Contracting Activity on Rat Stomach Strip (meacounter-current partition chromatography by employsurcd by the method of Magnus): ing a column having an inner capacity of 240 ml. More threshold dose (ll ng/ml 6i specifically. an upper layer of a liquid of ndose-response: observed butanolzacetic acid:water (411:5) was used as the sta- 2. Contracting Activity on Guinea Pig lleum (meationary phase, and the lyophilized product was dissured by the method of Magnus): solved in the water layer as the lower layer. The resulting aqueous solution was added dropwise to the stationary phase and the elution was effected at an elution rate of 3 ml/hr and at a unit fraction volume of 3 ml. Active fractions (48th to lth fractions) were collected, and concentrated under reduced pressure and dried to solids. The solids were dissolved in 0.5 ml of 0.001 N formic acid and the solution was charged in a column of a diameter of 0.9 cm and a length of 30 cm packed with SE-Sephadex C-25 bufferized sufficiently with a 0.] M ammonium formate liquor (pH 6.5). The de velopment elution was effected at an elution rate of [5 ml/hr and a unit fraction volume of 3 ml with use of 0.05 M ammonium formate (pH 6.5). Active fractions (47th to 63rd fractions) were collected and lyophilized to obtain 750 pg of a white powdery peptide.

This peptide was dansylated (dimethylaminonaphthalene-sulfonylated) and was subjected to the silica gel thin layer chromatography by employing two kinds of development solvents. namely a liquid of butanolzacetic acidzwater (4:l:5) and a liquid of methyl acetatezisopropanol:ammonia (9:7:4). As a result, it was confirmed that the obtained peptide was a single substance.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An octapeptide compound consisting of the following amino acids arranged in the following order and wherein the configurations of all the amino acids are L:

pyroglutamic acid.glycine.lysine.arginine.proline.

tryptophan.isoleucineleucine. 

1. AN OCTAPEPTIDE COMPOUND CONSISTING OF THE FOLLOWING AMINO ACIDS ARRANGED IN THE FOLLOWING ORDER AND WHEREIN THE CONFIGURATIONS OF ALL THE AMINO ACIDS ARE L: PYROGLUTAMIC ACID.GLYCINE.LYSINE.ARGININE.PROLINE. TRYPTOPHAN.ISOLEUCINE.LEUCINE. 